1. Field of the Invention
This invention is in the field of biotechnology. In particular, it relates to improvements in the post-transcriptional control of gene expression in eukaryotic cells.
2. Background of the Invention
Eukaryotic gene expression undergoes several points of control after transcription of primary mRNA from DNA. The primary mRNA transcript is comprised of coding portions (exons) and non-coding portions (introns). During mRNA splicing, introns are cut and removed from the transcript and exons are joined together to generate mature messenger RNA (mRNA). Splicing serves as a point of control for generating multiple protein isoforms from a single gene through the addition and removal of exons in various combinations. This process, termed alternative splicing, occurs within tightly regulated, multi-component structures called spliceosomes, which are under the control of intra- and extra-cellular signaling pathways.
Alternative splicing within the coding region of a protein can result in generation of multiple isoforms with diverse functions. Additionally, splicing has been shown to dramatically increase protein synthesis in mammalian cells (Huang and Gorman, 1990 Nucleic Acids Research 18(4):937-947). The mechanism for this is unknown. Alternative splicing can also occur in the untranslated regions of the transcript, which may contribute enhancer or stabilization domains to the final transcript, resulting in increased translation of protein.
Addition of splicing elements in the 5′ regulatory region in a synthetic gene construct has been shown to increase gene expression, theoretically as a result of improved mRNA transport from the nucleus to the cytoplasm (Huang and Gorman, supra; Choi et al., 1991 Molecular and Cellular Biology 11(6):3070-3074). As a result of this work, introns are often included between the promoter and multiple cloning site of commercially available mammalian expression vectors. However, combinations of introns with other regulatory regions have not been evaluated for increasing gene expression.